Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

• Images in the following figures have been replaced with unmodified versions, with no changes to contrast or brightness: Figs 4A (PS120 CD95(Δ1-210) panels), S9A and S10.
• Underlying image data for the western blot experiments in the following figures are provided in S1 File: Figs 2, 4, 5, S1, S5, and S8. The underlying blot for H9 cells in S1C Fig is no longer available.
The Materials and Methods section is supplemented with the following information provided in S2 File: • Protocol for the generation of control and CD95-expressing PS120-NHE1 cells.
• Protocol for the production of Ig-CD95.

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)." The corresponding author apologizes for the errors in the published article and previous Correction. ng/ml of cl-CD95L for 30 min. Cell morphology was followed using phase contrast microscopy and CD95 was stained using a mouse anti-CD95 mAb (APO1-3) followed by a goat Alexa488-coupled anti-mouse IgG mAb. Red arrows depict emitted pseudopods (Bars = 5 mm). (B) PHA/IL2-activated PBLs were incubated with the cytotoxic Ig-CD95L for indicated times, and cells were analyzed as described in (A). White arrows depict blebs emitted by apoptotic cells (bars = 5 mm). (C) The fibroblastic cell line PS120 devoid of endogenous CD95 (wild type NHE1-reconstituted PS120 control ) or reconstituted with human wild type CD95 (PS120 CD95 ) were untreated or treated with 100 ng/ml of cleaved CD95L for indicated times, and cell shape and CD95 distribution were analyzed. The "0" condition indicates cells analyzed before the addition of 100 ng/mL of cl-CD95L.
https://doi.org/10.1371/journal.pbio.3002027.g001 H9 T-cells were transiently transfected with the actin marker Lifeact-GFP. Living cells were harvested (Ficoll) and treated for 30 min with 100 ng/ml of cleaved CD95L or with control medium (supernatant of pcDNA3.1(+)-transfected HEK cells-untreated). Cells were fixed, and CD95 was stained using anti-CD95 mAb (APO1-3) and revealed with the secondary Alexa555-coupled Goat anti-mouse antibody (red). Nuclei were stained using DAPI (blue). Bars = 5 mm. Images were acquired with an ApoPLAN 63× objective. (B) The leukemic T-cell line H9 was incubated with 100 ng/ml of the naturally processed CD95L for indicated times. Cells were lysed, and for each lane, 100 mg of protein were loaded. Proteins were resolved by SDS-PAGE and antiwhole Akt and anti-Akt phosphorylation (serine 473 ) immunoblots were performed. Akt phosphorylation stands for its activation. (C) The pleckstrin homology (PH) domain of Akt binds PIP3 produced by PI3K activation. As a consequence, the probe PH Akt -GFP stains PIP3. H9 T-cells were transiently transfected with the PH Akt -GFP construct (green). Living cells were isolated (Ficoll) and incubated with 100 ng/ml of the naturally processed CD95L (cl-CD95L) or with control medium (supernatant from pcDNA3.1(+)-transfected HEK cells-untreated) for 30 min. Cells were fixed, and CD95 was stained as previously mentioned (red). Pictures were taken by confocal microscopy. Cell morphology was followed using differential interference contrast microscopy. Nuclei were stained with DAPI (blue). Bars = 5 mm. (D) Calcium measurement in single cell. Jurkat T-cells were loaded with the permeant calcium probe Fura-2AM, and in parallel, CD95 was followed using an anti-CD95 mAb and an Alexa555-coupled goat anti-mouse mAb as described in Materials and Methods. Cells were bathed in a Ca 2+ -free medium and pre-incubated (lower panel; cl-CD95L) or not (upper panel; supernatant from pcDNA3.1(+)-transfected HEK cells-untreated)) with cl-CD95L (100 ng/ml). 2 mM Ca 2+ was added when indicated by the black-filled rectangle. Ratio images (F340 nm/F380 nm) were taken every 5 s, and the images shown correspond to the indicated period of time. Grey level intensities were translated to false colors according to the colors scale shown at the right, and [Ca 2+ ]i was estimated from the ratio values and calibration experiments as described in Materials and Methods. Red arrows indicate the CD95-CAP. (E) Jurkat T-cells were treated with 100 ng/ml of cl-CD95L or with control medium (supernatant from pcDNA3.1(+)-transfected HEK cells-untreated) for 60 min. Cells were fixed and Orai1 and CD95 were stained as described in Materials and Methods. Cell morphology was analyzed using phase contrast microscopy. (F) Left panels: GFP-, GFP-Orai1, or GFP-Orai1 E106Aexpressing Jurkat T-cells were loaded with 1 mM of the calcium probe Fura-2AM for 30 min at RT. Cells were bathed at 37˚C in a medium containing 2 mM [Ca 2+ ]e and then treated with 100 ng/ml of cl-CD95L (black arrow). GFP#84 corresponded to control cells expressing GFP. GFP-Orai1 E106A #155 and #169 corresponded to two independently isolated clones expressing the non-functional CRAC channel. GFP-Orai1#142 corresponded to Jurkat cells overexpressing human Orai1. Values were recorded every 10 s. Right panel: For each experiment, the area under the curve (AUC) was measured for 1,000 s, and the statistical analyses of the AUC values were performed for indicated cells using non-parametric two-tailed Mann-Whitney tests. ��   The CD95-deficient PS120 (wild type NHE1-reconstituted PS120 control ) and its counterparts expressing human wild type CD95 or DD-truncated CD95 were seeded in a Boyden chamber in the presence of cl-CD95L (100 ng/ml) or with control medium (supernatant from pcDNA3.1(+)-transfected HEK cells-Untreated) and incubated for 24 h. The filter was removed, the upper side containing the non-migrating cells was wiped out with cotton-tipped swabs and migrating cells in the opposite side of the filter were fixed with methanol and stained (Giemsa). For each experiment, five pictures of random fields were taken, and a representative picture was depicted (Bars = 70 μm). (B) Cells were treated as described in (A). To quantify cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed and absorbance was measured at 560 nm. (C) Wound healing assay, a confluent monolayer of the indicated cells was "wounded" with a tip and then cells were incubated for 24 h in the presence of 100 ng/ml of cl-CD95L or a control medium (supernatant of pcDNA3.1(+)-transfected HEK cell) (untreated) and pictures were acquired (Bars = 50 μm).  The CD95-deficient fibroblastic cell line PS120 transfected with empty vector (wild type NHE1-reconstituted PS120 control ) or reconstituted with either human wild type CD95 (PS120 CD95 ) or a death domain-truncated CD95 (PS120 CD95(Δ1-210) ) was incubated with 100 ng/ml of cleaved CD95L for indicated times. Cells were harvested, lyzed, and 100 μg of protein was loaded per lane. Proteins were resolved in a SDS-PAGE and immunoblots were performed. Phosphorylation of the serine 473 on Akt indicates activation of the kinase. Whole Akt is added as loading control. The PS120 immunoblots shown in (A) have been partly reused in [3]. (B) Left panels: PS120 CD95 cells were pre-incubated with non-cytotoxic and non-cytostatic concentration of PI3K inhibitors (2.5 μM of wortmannin and 5 μM of LY294002), the cell permeant chelator of calcium BAPTA-AM (5 μM), the inhibitor of IP3-R and SOC channels 2-APB (20 μM) or DMSO (vehicle). Cells were then untreated (control medium from pcDNA3.1(+)-transfected HEK cells) or treated for 5 min with 100 ng/ml of cl-CD95L and lysed, 50 μg/ml of protein was loaded per lane and indicated immunoblots were performed. Right panel: the densitometry analyses of the immunoblot bands were performed using ImageJ software and the histograms depict Phospho-Akt/whole Akt ratios. (C) Cell migration of PS120 CD95 was assessed using the Boyden chamber assay. Cells were pre-incubated with non-cytotoxic and non-cytostatic concentrations of PI3K inhibitors (2.5 μM of Wortmannin and 5 μM of LY294002), the cell permeant chelator of calcium BAPTA-AM (5 μM), the inhibitor of SOC channels 2-APB (20 μM) or DMSO (vehicle) for 30 min and then stimulated with 100 ng/ml of cl-CD95L or a control medium (supernatant from pcDNA3.1(+)-transfected HEK cell-untreated) for 24 h. For each experiment, 10 pictures of the migrating cells were taken, and a representative picture was depicted (Bars = 50 μm). (D) Cells were treated as described in (C). To quantitatively measure cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed, and absorbance was measured at a wavelength of 560 nm. Values represent means and SD of three independently performed experiments. �� p�0.01 and ��� p�0.001 as calculated using two-tailed non-parametric Mann-Whitney test. (E) The silencing effect of the Orai1-targeting shRNAmir-pGIPZ vectors was analyzed by immunoblot in lentiviral-transduced HEK cells. 48 h after transduction, cells were lysed and 100 mg of lysates were loaded per line. β-actin serves as a loading control. (F) Cell migration of indicated shRNAmir-transduced HEK cells was assessed using Boyden chamber assay. Right panels: HEK cells were transduced with scrambled or Orai1-targeting ShRNA, and 48 h later, cells were incubated for 24 h in the presence of 100 ng/ml of cl-CD95L or untreated (supernatant of pcDNA3.1(+)-transfected HEK cells). Migrating cells were fixed with methanol and stained by Giemsa. For each experiment, five pictures of random fields were taken, and a representative picture was depicted (Bars = 70 mm). Left panel: To quantitatively measure cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed, and absorbance was measured at a wavelength of 560 nm. Values represent means and SD of three independently performed experiments. ��� p�0.001 as calculated using two-tailed non-parametric Mann-Whitney test.
https://doi.org/10.1371/journal.pbio.3002027.g004 The leukemic cell line H9 was transiently transfected with Lck-GFP. 24 h after transfection, living cells were treated or untreated for the indicated times in the presence of cl-CD95L (100 ng/ml). 0 min (0') condition corresponds to cells analyzed prior to the addition of 100 ng/mL of cl-CD95L. CD95 was stained using a mouse anti-CD95 mAb and a goat Alexa555-coupled anti-mouse IgG mAb. Right panels: Activated PBLs were incubated for the indicated times with 100 ng/ml of cl-CD95L, and then cells were fixed. Lipid rafts were stained using Alexa488-cholera toxin B subunit and CD95 was followed as previously mentioned. Images were acquired with a confocal microscope with a 63× objective. 0 min (0') condition corresponds to cells analyzed prior to the addition of 100 ng/mL of cl-CD95L. Cell morphology was followed using phase contrast microscopy (Bars = 7.5 mm). The percentage of T-cells displaying a CD95 cluster was assessed (300 cells counted for each condition). Among the activated T-lymphocytes showing CD95 cluster, the amount of CD95-CAP co-localized with lipid rafts was assessed. (B) H9 T-cells were preincubated for 30 min with DMSO (vehicle), 10 mM of PP2, 5 mM of LY294002 (LY), 2.5 mM of wortmannin (Wort), or 40 mM of zVAD-fmk (zV) and then treated for 15 min with 100 ng/ml of cl-CD95L. Untreated condition corresponds to cells incubated with supernatant of pcDNA3.1(+)-transfected HEK cells. Cells were lyzed and Akt phosphorylation (S 473 ) and whole Akt were assessed by immunoblots. (C) The H9 T-cell line (left panels) and activated PBLs (right panels) were incubated for indicated times with 100 ng/ml of cl-CD95L or APO1-3. APO1-3 is an agonistic anti-CD95 mAb incubated for 15 min with indicated cells to activate CD95 and trigger DISC formation. Then, cells were lyzed and CD95 was immunoprecipitated. The 0 min condition corresponds to unstimulated cells directly lyzed to next immunoprecipitate CD95. APO1-3 also serves for the immunoprecipitation step of CD95 (see Materials and Methods). The immune complex was resolved in a 10% SDS-PAGE and indicated immunoblots were performed. Total lysates were depicted to confirm that the same amount of protein was present for each immunoprecipitation. The black stars represent heavy chains of Ig. (D) Boyden chamber assays were performed as described in Materials and Methods. PS120 CD95 (All PS120 cells have been reconstituted with wild type NHE1 (see Materials and Methods) were pre-incubated with or without a non-cytotoxic and non-cytostatic concentration of PP2 (10 mM . PS120 control cells do not express CD95, while its counterpart PS120 CD95 expresses a full length human CD95 (see Materials and Methods). A 24 h wound healing assay was performed. The fibroblastic cell line PS120 control or its CD95-expressing counterpart PS120 CD95 was grown to confluence. then the monolayer was "wounded" by scratching cells using a pipette tip. Using microscopy, the migration was monitored for 24 h with the indicated concentrations of cl-CD95L (ng/mL) as The leukemic T-cell line H9 was incubated for indicated times with 100 ng/ ml of cleaved CD95L or a control medium (supernatant of pcDNA3.1(+)-transfected HEK cells-untreated), and after extensive washing, cells were fixed. CD95 was stained using an anti-CD95 mAb (APO1-3) and revealed using the secondary Alexa488-conjugated goat antimouse antibody (Invitrogen, Carlsbad, CA, USA). Number of cells harboring a CD95-Cap was counted (at least 300 cells counted for each condition). (B) The leukemic T-cell lines CEM and H9 were incubated for 30 min with 100 ng/ml of cleaved CD95L or a control medium (supernatant of pcDNA3.1(+)-transfected HEK cells -untreated), and the plasma membrane distribution of CD95 was then analyzed as described in (A). The quantity of cells harboring a CD95-Cap was assessed by counting (300 cells/condition). Values represent means and SD of three independently performed experiments. �� p<0.01 and ��� p<0.001 as calculated using non-parametric and two-tailed Mann-Whitney test. (C) Leukemic T-cells H9 and CEM were untreated (supernatant of pcDNA3.1(+)-transfected HEK cells) or treated for 30 min with cleaved CD95L (100 ng/ml), and after extensive washing, cells were fixed. CD95 was stained using an anti-CD95 mAb (APO1-3) and revealed using a secondary Alexa488-conjugated goat anti-mouse antibody. Nuclei were stained with DAPI (blue). Slides were washed with PBS, dried, and mounted with Fluoromount (Cliniscience SAS, Montrouge, France). Red arrows depict emitted pseudopods upon CD95 engagement. Images were acquired with a confocal microscope TSC SP5 (Leica, Wetzlar, Germany) with a ApoPLAN 63× objective. (TIF) S3 Fig. CD95 and truncated CD95(1-210)-expressing PS120 clones. All PS120 cells are reconstituted with wild type NHE1 (see Materials and Methods). (A) Upper panel: analysis of the CD95 expression at the surface of the indicated PS120 clones stably expressing either the CD95 wild type or its death domain truncated counterpart (Δ1-210). Cells were stained with an anti-CD95 mAb (clone DX2), washed, and a PE-coupled goat anti-mouse secondary antibody was used to reveal plasma membrane CD95 by flow cytometry. Lower panel: for each staining, the mean of the fluorescence intensity (MFI), which is correlated to the amount of plasma membrane CD95, was depicted. FACS analyses of PS120 cells shown in (A) have been partly reused in [3]. (B) Indicated PS120 clones expressing either empty vector (PS120 control ), truncated (PS120 CD95(Δ1-210) ), or wild type CD95 (PS120 CD95 ) were incubated with the cytotoxic Ig-CD95L or the cleaved CD95L for 24 h and cell death was quantified using viability assay MTT. (TIF)

S4 Fig. Cleaved CD95L triggers a pseudopod-localized Ca 2+ rise. Video imaging on living cells:
Before application of cl-CD95L, activated T-lymphocytes were loaded with the Ca 2+ indicator Fura-2AM along with EGTA-AM (1 mM, 30 min, room temperature), a slow high-affinity Ca 2+ buffer. Under these conditions, Ca 2+ entering the cell would bind rapidly to the Ca 2+ probe, producing a fluorescent signal, and then be captured by EGTA. Activated T-lymphocytes were bathed in an external medium containing 2 mM Ca 2+ and stimulated with 100 ng/ ml of cl-CD95L just after the capture of the ratio images (0 s). Pictures were recorded at the indicated times following the addition of cl-CD95L to assess the formation of localized Ca 2+ influx. Emitted pseudopods were depicted using a white square, and cell migration is indicated by a white arrow. Grey levels were translated to false colors according to a scale shown on the right. CD95-expressing PS120 cells were pre-incubated with the pan-caspase inhibitor zVAD-fmk (40 μM) or DMSO (vehicle) for 30 min and then seeded in the upper compartment of the Boyden chamber in a low FCS (1%)-containing medium. Cells were incubated for 24 h with 100 ng/ml of cl-CD95L or with a supernatant of pcDNA3.1(+)-transfected HEK cells (untreated). Then the membrane was removed, the upper side containing the non-migrating cells was wiped out with cotton-tipped swabs, and migrating cells in the opposite side of the filter were fixed with methanol and stained by Giemsa (purple cells). For each experiment, five pictures of random fields were taken and a representative picture was depicted (Bars = 70 μm). The images of PS120 CD95 cells untreated/vehicle and cl-CD95L/vehicle came from the same experiment to decipher the effect of PP2 (src inhibitor) and zVAD (caspase inhibitor) and were used in S9B and 5D Figs. Lower panel: To quantify cell motility, Giemsa-stained migrating cells from the lower side of the membrane were lyzed and absorbance was measured at 560 nm.